Research On The Influence Of Neuro-immunodulation Based On Substance P And Calcitonin Gene Related Peptide On Mouse Skin Allograft Survival

Kidney transplantation is one of the most efficient methods to cure end stage renal disease, however, there is still some unsolving problems hindering the development of kidney transplantation, such as a variety of side effects and adverse reactions of immunosuppressants, immunosuppression related infections of the recipients, individual differences of the absorption and metabolation of immunosuppressants, and in some patients, the therapeutic window is very narrow, which may lead to infection and rejection coexist in one case of patient.Calcineurin inhibitors are the cornerstone of triple immunosuppressive regimen, however, it’s drawbacks are innegligible, it causes chronic neprotic toxicity, which was responsible for the declination of the graft long term survival rate. Most of researchers and clinicians are making great efforts in the field of organ transplantation, trying to find a ideal solution of such problems. This study focuses on neuroimmunodulation in allotransplantation.Nervous system and immune system is inseparable. In the course of neurogenic inflammation, substance P (SP) and calcitonin gene related peptide (CGRP), which are two of the most important neuropeptides, are released by nerve endings. Neurogenic inflammation can be triggered by neuropeptides and expanded by nervous networks, axon reflex and dorsal root reflex play a key role in the pathgenesis of inflammation expansion. SP and CGRP have a synergistic effect in neurogenic inflammation. Specific neuropeptide receptors are expressed on the surface of immune cells, which can be binded with corresponding neuropeptides and regulate the function of immune cells. The collaborative neuropeptides, SP and CGRP, have reverse reaction in immune responses:SP promotes T cell proliferation while CGRP inhibit the activation of T cells.As ervous network is widespread in the body, neuroimmunodulation have the potential of assimilate the immune states of massive immune cells in a short time. As above mentioned, SP and CGRP have reverse reaction in immune responses,which suggests that immunodulation based on the level of neuroimmune may be a very important regulate point. Neuroimmunodulation don’t influence to immune cells directly, it may have paiticular advantages compared with traditional immunodulations. However, if neuroimmunodulation is fully efficient to protect allograft is unknown. This study will build mice skin transplantation model and focus on the influence of this immunodulation method on skin graft survival, balance of Th1and Th2cell subsets, formation of Tregs and expression of critical molecules on the signaling pathways of lymphocyte activation.Part Ⅰ Building of mice allogene skin transplantation modelObjective:To successfully build mice allogene skin transplantation model.Methods:Full thickness skin grafts were prepared, BALB/c mice accepted allogene skin transplantation and autogenous skin transplantation respectfully, survival condition of the skin graft was observed and recordered.Results:On the4th day posttransplantation, the allograft was smooth on the surface and turned red, there was no floating phenomenon. On the7th day posttransplantation, the allograft was rejected, the graft shrank and became rigid, dry necrosis was observed on the graft, with dark crust looking. On the6th day posttransplantation, the autogenous skin graft was smooth and turned red, there was no floating phenomenon, the skin graft didn’t shrink during the course of fixation, On the12th day posttransplantation, the graft healed firmly with good flexibility, hair reappeared on the graft. When the suture was removed, the graft shranked40-50%, and keeped stable thereafter.Conclusion:1. Mice allogene full thickness skin transplantation was successfully built.2. This model is ideal for the research of allogene rejection and corresponding modulations.3. Neurogenic inflammation will be inevitably induced by surgical operation, this model is hence an ideal object to perform neuroimmunodulation. Part Ⅱ Influence of neuroimmunodulation on the survival of mice allograftObjective:Based on the successfully built mice skin transplantation model, different neuropeptide receptor antagonists was injected in the body of recipient mice, survival time of the skin graft was observed and compared in different groups.Methods:The mice accepted allotransplantation and autogenous transplantation were divided into different subgroups respectively, natural saline (NS), SP receptor antagonist Spantide, GCRP receptor antagonist BIBN4096BS and Spantide+BIBN4096BS were used as intervention, survival time of the skin graft was compared in these groups using Kaplan-Meier method.Results:Observation time was25days. The average survival time of autogenous skin graft was25days, the average survival time of allogenic skin graft was7days in NS subgroup,13days in Spantide subgroup,12days in BIBN4096BS subgroup and19days in Spantide+BIBN4096BS subgroup respectively.Conclusion:1. Both SP and CGRP immunodulation can prolong the survival time of mice’s allogenic skin graft.2. There is no difference in the survival time of mice’s allogenic skin graft between SP group and CGRP group.3. SP/CGRP comodulation can further prolong the the survival time of mice’s allogenic skin graft, suggests that SP and CGRP also have a synergistic effect in the level of neuroimmunodulation.4. Compared to conventional ways of immunodulation, the effect of neuroimmunodulation based on SP/CGRP is limited in prolonging the the survival time of mice’s allogenic skin graft, however, it is a promising research area. Part Ⅲ Immunohistochemistry analysis of SP/CGRP and fluorescence quantitative PCR of SP mRNA/CGRP mRNA in mice’s skin graftObjective:To qualitatively and quantitatively analyze SP and CGRP in mice’s skin graft on the level of peptide and gene expression.Methods:1. SP and CGRP in the mice’s skin graft were immunohistochemistrily stained.2. SP/CGRP mRNA in mice’s skin graft was analyzed by fluorescence quantitative PCR.3. The results of fluorescence quantitative PCR were analyzed by SPSS18.0, Mean comparison in groups was conducted with variance analysis, and the pairwise comparison was performed with LSD method, comparison between allo-group and autogenous group was performed with t test.Results:1. Immunohistochemistry revealed that the autogenous skin graft survived100%, epidermis had normal thickness, skin appendages can be observed. Epidermis of allogenic skin graft was found thinning out and skin appendages can not be found.2. Both SP and CGRP antagonism inhibit the expression of SP and CGRP, SP+CGRP co-modulation make such inhibition more significant.3. Allogenic transplantation mice and autogenous transplantation mice accept the same intervention were compared, expression of CGRP in allogenic transplantation group was significant lower than that in autogenous transplantation group.4. Skin tissue SP mRNA expression in autogenous transplantation mice were compared between different intervention groups, expression of SP mRNA in Ctrl②group was found significantly higher than that of SP②group and SP+CGRP②group, there was no significant difference when compared with CGRP②group. Expression of SP mRNA in SP②group was significant lower than that in CGRP②group, there was no significant difference when compared with SP+CGRP②group. Expression of SP mRNA was significantly higher than that in SP+CGRP②group.5. Skin tissue SP mRNA expression in allogenic transplantation mice were compared between different intervention groups, expression of SP mRNA in Ctrl①group was significantly than that in SP①group, group and SP+CGRP①group. Expression of SP mRNA in group was significantly lower than that in CGRP①group and SP+CGRP①group. Expression of SP mRNA in CGRP①group was significantly higher than that in SP+CGRP①group.6. Skin tissue CGRP mRNA expression in autogenous transplantation mice were compared between different intervention groups, expression of CGRP mRNA in Ctrl②group was significantly higher than that in CGRP②group, there was no significant difference when compared with SP②group and SP+CGRP②group. Expression of CGRP mRNA in SP②group was significantly higher than that in group and SP+CGRP②group. Expression of CGRP mRNA in group was significantly higher than that in SP+CGRP②group.7. Skin tissue CGRP mRNA expression in allo genic transplantation mice were compared between different intervention groups, expression of CGRP mRNA in Ctrl①group was significantly higher than that in CGRP①group and SP+CGRP①group, there was no significant difference when compared with SP①group. Expression of CGRP mRNA in SP①group was significantly higher than that in CGRP①group and SP+CGRP①group. There was no significant difference of the expression of CGRP mRNA between CGRP①group and SP+CGRP①group. 8. mice accepted the same intervention were chosen, their skin tissue SP mRNA expression was compared between allogenic and autogenous group, expression of SP mRNA in NS injected autogenous transplanted mice was significant lower than that in NS injected allogenic transplanted mice. Expression of SP mRNA in Spantide injected autogenous transplanted mice was significant higher than that in Spantide injected allogenic transplanted mice. There was no significant difference of the expression of SP mRNA between BIBN4096BS injected autogenous transplanted mice and BIBN4096BS injected allogenic transplanted mice. Expression of SP mRNA in Spantide+BIBN4096BS injected autogenous transplanted mice was significant lower than that in allogenic transplanted mice.9. mice accepted the same intervention were chosen, their skin tissue CGRP mRNA expression was compared between allogenic and autogenous group, expression of CGRP mRNA in NS injected autogenous transplanted mice was significant lower than that in NS injected allogenic transplanted mice. Expression of CGRP mRNA in Spantide injected autogenous transplanted mice was significant lower than that in Spantide injected allogenic transplanted mice. Expression of CGRP mRNA in BIBN4096BS injected autogenous transplanted mice was significant higher than that in BIBN4096BS injected allogenic transplanted mice. Expression of CGRP mRNA in Spantide+BIBN4096BS injected autogenous transplanted mice was significant higher than that in allogenic transplanted mice.Conclusion:1. In mice’s skin transplantation model, surgical operation was the initiative factor of neurogenic inflammation, secondary neurogenic inflammation can be self reinforced and promote neuroimmune reaction.2. Single blocked of SP receptor not only influence the expression and formation of SP, but also influence the expression and formation of CGRP, the same phenomenon was observed in CGRP receptor single blocked mice, which indicate SP and CGRP have cross feedback and synergistic effect.3. SP+CGRP co-blocked can further decrease the expression of SP and CGRP, but was not fully inhibition, which maybe a very important defect of neuroimmunodulation, results in a very limited survival time of allogenic graft in our study.4. An unknown immune reinforce mechanism based on the level of neuroimmune may be activated when the body of the mice were faced with allogenic grafts, which will accelerate the damage of the graft, SP+CGRP co-blocked can efficiently alleviate this affect.5. In the course of neurogenic inflammation, production of neurupeptides and expression of their mRNA do not always display strict linear relationship.6. Allogenic immune response can significantly promote the expression of SP mRNA in skin graft than neurogenic inflammation. Part IV Testing of Thl cytokins and Th2Cytokins in peripheral blood of skin transplanted miceObjective:To test Th1cytokins IFN-y, IL-2and Th2cytokins IL-10in peripheral blood of skin transplanted mice, indirectly evaluate the influence of neuroimmunodulation on the mice’s immune system.Methods:1. ELISA technique was used to quantitatively test Th1cytokins IFN-y, IL-2in mice’s peripheral blood.2. ELISA technique was used to quantitatively test Th2cytokins IL-10in mice’s peripheral blood.3. The results were analyzed by SPSS18.0, Mean comparison in groups was conducted with variance analysis, and the pairwise comparison was performed with LSD method, comparison between allo-group and autogenous group was performed with t test.Results:1. Neuroimmunodulation decreased the expression level of IFN-yin autogenous/allogenic transplanted mice’s peripheral blood, the extent of influence was in sequence of SP+CGRP co-antagonism> CGRP antagonism> SP antagonism> NS.2. Neuroimmunodulation decreased the expression level of IL-2in autogenous/allogenic transplanted mice’s peripheral blood, the extent of influence was in sequence of SP+CGRP co-antagonism> CGRP antagonism> SP antagonism> NS.3. Neuroimmunodulation promoted the expression level of IL-2in autogenous/allogenic transplanted mice’s peripheral blood, the extent of promotion was in sequence of SP+CGRP co-antagonism> CGRP antagonism> SP antagonism> NS.4. mice accepted the same intervention were chosen, their peripheral blood cytokins were compared between allogenic and autogenous group, expression level of IFN-y, IL-2, IL-10in allogenic group was significantly than that in autogenous group, this kind of significane exists in most of the intervention groups.Conclusion:1. By analyzing the results of Thl cytokins and Th2cytokins, we can conclude that neuroimmunodulation based on SP and CGRP can play protective role on mice’s allogenic skin graft, the extent of protection was in sequence of SP+CGRP co-antagonism> CGRP antagonism> SP antagonism.2. Changing trend of Thl and Th2cytokins in autogenous transplanted mice was similar to that of allogenic transplanted mice, which indicate that the influence of neuroimmunodulation on immune system do not rely on allogenic immune reaction.3. The influence of CGRP antagonism on allogenic transplanted mice’sThl cytokins was greater than that in autogenous transplanted mice, however, there was no significant difference in the expression level of Th2cytokins between the two groups, it may be related to the declined expression of GCRP in allogenic skin transplantation mice, that was why we found the protective effect of CGRP antagonism was greater than that of SP antagonism. Part V Quantitative analysis of critical signaling molecules on mice’s lymphocyte activation pathwaysObjective:To quantitatively analyze critical signaling molecules on3main lymphocyte activation pathways in skin transplanted mice. Find the way neuroimmunodulation influence mice’s immune system.Methods:1. Fluorescence quantitative PCR was used to quantitatively analyze the expression of critical signaling molecules in MAPK pathway.2. Fluorescence quantitative PCR was used to quantitatively analyze the expression of critical signaling molecules in Calcineurin/NFAT pathway.3. Fluorescence quantitative PCR was used to quantitatively analyze the expression of critical signaling molecules in JAX-STAT pathway.4. The results were analyzed by SPSS18.0, Mean comparison in groups was conducted with variance analysis, and the pairwise comparison was performed with LSD method, comparison between allo-group and autogenous group was performed with t test.Results:1.4critical signaling molecules in MAPK pathways were quantitatively analyzed, neuroimmunodulation based on SP/CGRP was found to have on influence on the expression of ERK gene. The influences on JNK2gene may be main related to inflammation. Infuences of neuromoduation on JNK1gene and P38MAPK gene were opposite: SP modulation promoted P38MAPK gene expression in autogenous transplanted mice, but inhibit JNK1gene expression in allogenic transplanted mice, CGRP modulation inhibited P38MAPK gene expression in allogenic transplanted mice, but promote JNK1gene expression in autogenous transplanted mice.2.2critical signaling molecules in Calcineurin/NFAT pathways were quantitatively analyzed, neuroimmunodulation was found can promote NFAT gene and Calcineurin gene expresison, NFAT gene expression can be relatively strongly induced by SP modulation, Calcineurin gene expression can be relatively strongly induced by CGRP modulation.3.2critical signaling molecules in JAK/STAT pathways were quantitatively analyzed, neuroimmunodulation was found to have on influence on the expression of both JAK1gene and Tyk2gene.Conclusion:1. Neuroimmunodulation based on SP/CGRP has on influence on the expression of ERK gene and JNK2gene. Infuences of neuromoduation on JNK1gene and P38MAPK gene were opposite:SP modulation promoted P38MAPK gene expression in autogenous transplanted mice, but inhibit JNK1gene expression in allogenic transplanted mice, CGRP modulation inhibited P38MAPK gene expression in allogenic transplanted mice, but promote JNK1gene expression in autogenous transplanted mice.2. Neuroimmunodulation based on SP/CGRP can both promote the expression of Calcineurin gene and NFAT geng in Calcineurin/NFAT pathway, NFAT gene expression can be relatively strongly induced by SP modulation, Calcineurin gene expression can be relatively strongly induced by CGRP modulation.3. Neuroimmunodulation based on SP/CGRP had no influence on the expression of JAK1gene, it almost had no obvious influence on Tyk2gene in allogenic transplanted mice. Part VI Testing of CD4+CD25+Treg and Foxp3in skin transplanted mice’s peripheral bloodObjective:To investigate if neuroimmunodulation is efficient enough to induce CD4+CD25+Treg formation.Methods:1. Fluorescence quantitative PCR was used to quantitatively analyze the expression of Foxp3mRNA in mice’s skin graft tissues.2. Flow cytometry was used to separate and test CD4+CD25+Tregs in mice’s peripheral blood. 3. The results of fluorescence quantitative PCR were analyzed by SPSS18.0, the results of flow cytometry were analyzed by BD FACS CantoTMⅡ. Mean comparison in groups was conducted with variance analysis, and the pairwise comparison was performed with LSD method, comparison between allo-group and autogenous group was performed with t test.Results:1. The percentage of CD4+CD25+Foxp3+Tregs in neuroimmunodulated mice’s peripheral blood rises, the influence degree to Tregs was SP modulation> SP+CGRP co-modulation> CGRP modulation>NS control.2. mice accepted the same intervention were compared between allogenic/autogenous teams, the percentage of CD4+CD25+Foxp3+Tregs in autogenous transplanted mice’s peripheral blood was significantly lower than that in allogenic transplanted mice’s peripheral bloods.3. The results of Foxp3quantitation was unidentical with flow cytometric results, which may because of the specimen were obtained from different resources.Conclusion:1. Neuroimmunodulation induce the formation of CD4+CD25+Foxp3+Tregs in mice’s peripheral blood.2. The influence degree of neuroimunodulation to Tregs was SP modulation> SP+CGRP co-modulation> CGRP modulation> NS control.